Journal: Molecular Cancer

Article Title: Systemic immunosuppression limits NK cell therapy efficacy in pancreatic cancer

doi: 10.1186/s12943-025-02340-8

Figure Lengend Snippet: Depletion of Gr-1+ cells rescues the effector functions of splenic NKs in PDAC mice. A Percentage of Tregs (CD3+, CD4+, CD25+, FOXP3+) and NKs (CD3-, NK1.1+) in the spleens of healthy and KPC1245 tumor-bearing mice (PDAC mice) treated with anti-CD25 antibody or isotype control. B Percentage of Dying B16F10 cells following co-culture with splenic NK (E: T = 20:1, 20 h) from healthy or PDAC mice treated with anti-CD25 antibody or isotype control. C Expression of IFNγ and GZMB in splenic NKs from different cohorts (as described in panel B) after stimulation with B16F10 for 20 h. D Percentage of macrophages (CD11b+, F4/80+, CD11c-) and MDSCs (CD11b+, Gr-1+, CD11c-) in the spleen of healthy and PDAC mice treated with anti-Gr-1, anti-CSF1R, or isotype control antibodies. E Percentage of dying KPC1245, YAC1, and B16F10 cells following co-culture with splenic NKs from healthy and PDAC mice treated with anti-Gr-1, anti-CSF1R antibodies, or isotype control. F Expression of IFNγ, GZMB, and CD107a in splenic NKs from different mouse cohorts upon stimulation with target cells. G Percentage of dying YAC-1 and B16F10 cells following co-culture with NKs pre-incubated with or without splenic MDSCs isolated from PDAC mice. CTL NK: healthy splenic NKs; NK co-Gr-1+ cells: healthy splenic NKs were pre-incubated with PDAC splenic Gr-1+ (MDSCs). H Expression of IFNγ, GZMB, and CD107a in control and Gr-1+ cells-co-cultured splenic NKs from different treatment cohorts after stimulation with or without B16F10 cells. Data are presented as mean +/- SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA with Tukey’s multiple comparisons test was used for panels A-E, &G; Two-way ANOVA with Tukey-Kramer test was used for panels F &H. The stars without bars represent the comparison between the first column and the column marked with stars

Article Snippet: NKs and Gr-1+ cells were isolated using an NK cell isolation kit (Miltenyi Biotec, Cat# 130-115-818) and an MDSC isolation kit (Miltenyi Biotec, Cat# 130-094-538).

Techniques: Control, Co-Culture Assay, Expressing, Incubation, Isolation, Cell Culture, Comparison

Journal: Molecular Cancer

Article Title: Systemic immunosuppression limits NK cell therapy efficacy in pancreatic cancer

doi: 10.1186/s12943-025-02340-8

Figure Lengend Snippet: PDAC splenic Gr-1+ cells induce lipid accumulation and lipid peroxidation in NKs. A Mean fluorescent intensity (MFI) of BODIPY staining in splenic NKs from healthy and KPC1245 tumor-bearing mice (PDAC mice) treated with isotype control or anti-Gr-1 antibody. B MFI of BODIPY in NKs co-incubated for 48 h without or with splenic MDSCs from PDAC mice. C Relative ratio of C11-BODIPY (lipid ROS measurement) and DCFDA intensity in splenic NKs from healthy and PDAC mice treated with isotype control or anti-Gr-1 antibody. D Relative DCFDA intensity in NKs co-incubated for 48 h without or with splenic Gr-1+ (MDSCs) from KPC1245 tumor-bearing mice. E Relative ratio of C11-BODIPY MFI in NKs co-incubated without or with splenic MDSCs from PDAC mice. F & G Relative DCFDA intensity (F) and the ratio of C11-BODIPY MFI (G) in splenic NKs co-incubated with MDSCs from PDAC mice and treated without or with vitamin E (VitE). H Percentage of dying YAC-1 and B16F10 cells following co-culture with splenic NKs co-incubated with MDSCs from PDAC mice and treated without or with vitamin E (VitE). I Expression of IFNγ, GZMB, and CD107a in splenic NKs from different cohorts (as shown in panel H) after stimulation without or with B16F10 cells. J–L MFI of BODIPY, DCFDA, and the C11-BODIPY relative ratio in NKs from the spleen of healthy and PDAC mice treated without or with VitE. M Percentage of dying KPC1245, YAC-1, and B16F10 cells following co-culture with splenic NKs from healthy mice or PDAC mice treated without or with VitE. N Expression of IFNγ in splenic NKs from different cohorts (as shown in panel M) after stimulation with B16F10 cells. Data are shown as the mean +/- SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA with Tukey’s multiple comparisons test was performed for panels A, C, and F-N. Unpaired Student’s t-test (two-tailed) was performed for panels B, D, and E. The stars without bars represent the comparison between the first column and those marked with stars.

Article Snippet: NKs and Gr-1+ cells were isolated using an NK cell isolation kit (Miltenyi Biotec, Cat# 130-115-818) and an MDSC isolation kit (Miltenyi Biotec, Cat# 130-094-538).

Techniques: Staining, Control, Incubation, Co-Culture Assay, Expressing, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: Systemic immunosuppression limits NK cell therapy efficacy in pancreatic cancer

doi: 10.1186/s12943-025-02340-8

Figure Lengend Snippet: APOE is essential for the suppression of NK cell function mediated by myeloid cells. A GSEA reveals significant alterations in lipid metabolic pathways in single-cell clusters from human PDAC compared to normal pancreases (scRNA-seq data from Peng et al., 2019). B Myeloid cells from PDAC patients exhibit a distinct gene signature score for lipid metabolic pathways compared to healthy individuals (scRNA-seq data from Steele, N.G. et al., 2020). C Top 20 lipid metabolism-related genes differentially expressed in myeloid cells from PDAC tumors compared to normal controls (scRNA-seq data from Peng, J. et al., 2019; Steele, N.G. et al.,2020). D mRNA expression profiles of the four commonly altered genes identified in panel C across 12 major cell types. E & F Percentage of dying YAC-1 and B16F10 cells after co-culture with splenic NKs (E: T = 20:1, for 20 h) from different pre-cultured cohorts. CTL NK, healthy splenic NK cells; NK-coMDSCs WT: splenic NK cells pre-incubated with splenic Gr-1 ± (MDSCs) from wild-type PDAC mice for 48 h; NK-coMDSCs Apoe KO: splenic NK cells pre-incubated with Apoe -knockout MDSC isolated from Apoe -knockout PDAC mice for 48 h. G–I Expression of IFNγ, GZMB, and CD107a in healthy splenic NKs post-stimulation with or without B16F10 was assessed after pre-culture with splenic WT or Apoe -KO MDSCs as described in panel E &F. J–L Mean fluorescent intensity (MFI) of BODIPY staining (J), relative DCF intensity (K), relative ratio of C11-BODIPY MFI (L) in healthy splenic NKs after co-incubation with WT or Apoe- knockout splenic Gr-1 ± (MDSCs) isolated from PDAC mice. Data are shown as the mean +/- SD. One-way ANOVA with Tukey’s multiple comparisons test was used for panel E , F , and J–L Two-way ANOVA with Tukey-Kramer test was used for panel G-I. * p < 0.05, ** p < 0.01, and *** p < 0.001. The stars without bars represent the comparison between the first column and the column marked with stars

Article Snippet: NKs and Gr-1+ cells were isolated using an NK cell isolation kit (Miltenyi Biotec, Cat# 130-115-818) and an MDSC isolation kit (Miltenyi Biotec, Cat# 130-094-538).

Techniques: Cell Function Assay, Single Cell, Expressing, Co-Culture Assay, Cell Culture, Incubation, Knock-Out, Isolation, Staining, Comparison

Journal: Molecular Cancer

Article Title: Systemic immunosuppression limits NK cell therapy efficacy in pancreatic cancer

doi: 10.1186/s12943-025-02340-8

Figure Lengend Snippet: Disruption of lipid oxidation impairs the immunosuppressive effect of Gr-1+ cells on NKs. A Potential targets for blocking fatty acid oxidation (FAO) in Gr-1+ cells (MDSCs) to rescue NK cell function. B Heatmap showing the average expression of the genes related to lipid degradation in myeloid cells from peripheral blood mononuclear cells (PBMC) and PDAC tumors (scRNA-seq data from Peng, J. et al., 2019, Steele, N.G. et al., 2020, and Sun, D. et al., 2021). C–E The ratio of MDSCs in the spleen (C), the percentage of Arginase-1 and IL10 positive splenic MDSCs (D), and the relative DCFDA intensity in splenic MDSCs (E) were assessed in healthy control (HC) and KPC1245 pancreatic tumor-bearing mice (PDAC mice) treated with vehicle control (CTL), Orlistat (OLS), Trimetazidine (TMZ), and BMX-001 (BMX). F–K The percentage of NKs in the spleens (F), relative DCFDA intensity in splenic NKs(G), relative green/red ratio of C11-BODIPY fluorescence in splenic NKs (H), and the percentage of PD-1, LAG3, and TIGIT positive splenic NKs (I-K) were assessed in healthy and PDAC mice treated with CTL, OLS, TMZ, and BMX. L–N Percentage of dying KPC1245, YAC-1, and B16F10 cells after co-culture with splenic NKs (E: T = 20:1, for 20 h) from healthy and PDAC mice treated with CTL, OLS, TMZ, and BMX. O Expression of IFNγ, GZMB, and CD107a in splenic NKs (from panel N) after stimulation with B16F10 for 20 h. Data is presented as median. One-way ANOVA with Tukey’s multiple comparisons was performed for panels C-O. * p < 0.05, ** p < 0.01, and *** p < 0.001. The stars without bars represent the comparison between the first column and the column marked with stars

Article Snippet: NKs and Gr-1+ cells were isolated using an NK cell isolation kit (Miltenyi Biotec, Cat# 130-115-818) and an MDSC isolation kit (Miltenyi Biotec, Cat# 130-094-538).

Techniques: Disruption, Blocking Assay, Cell Function Assay, Expressing, Control, Fluorescence, Co-Culture Assay, Comparison